Flow cytometry is a technique for analyzing particles in a fluid, as will be understood by those skilled in the art. Briefly, particles are hydrodynamically focused in a stream of fluid. An optical energy source, such as a laser, is directed toward the fluid stream, and one or more detectors are positioned to receive scattered light from the particles, light emitted from fluorescent tags on the particles, or both. The detectors may be positioned both inline with the optical energy source, and at various angles to the optical energy source. Different detectors may be sensitive to different wavelengths of optical energy. By analyzing the signals received from the detectors, information about the physical or chemical properties of the particles may be determined. Histograms and other graphical plots may be generated that may depict particle counts or concentrations of particles containing the fluorescent tags.
Flow cytometry has been used to monitor cell signaling events. For example, a cellular immune response may encompass a variety of cellular events. Phosphorylation and dephosphorylation of intracellular proteins may play a vital role in signaling cascades. Measurement of these types of transitory signaling events may shed light on the timing and degree of immune response. Accordingly, flow cytometry may be used to measure an amount of phosphorylated protein. In particular, phospho-epitope staining has been used to stain cells, labeling the phosphate. In this manner, protein phosphorylation may be measured using flow cytometry techniques.
In order to utilize flow cytometry to monitor a cell signaling event, a number of sample preparation and handling steps may be necessary, including stimulation of the cells with a selected challenge to initiate a cell signaling event. A cell fixation step may then be performed to kill the cells and preserve or “freeze” the cell during a particular time in the cell signaling process. A permeabilization step may then be performed to make the cell membrane permeable. Then, phospho-specific antibodies are introduced into the cell to perform staining. Finally, a variety of washing and buffer steps may be required. Each step proceeds using its own respective lab equipment such as Petri dishes, pipettes, and centrifuge tubes.